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• W2044974504 abstract "Human AP endonuclease 1 (APE1, REF1) functions within the base excision repair pathway by catalyzing the hydrolysis of the phosphodiester bond 5 ′ to a baseless sugar (apurinic or apyrimidinic site). The AP endonuclease activity of this enzyme and two active site mutants were characterized using equilibrium binding and pre-steady-state kinetic techniques. Wild-type APE1 is a remarkably potent endonuclease and highly efficient enzyme. Incision 5 ′ to AP sites is so fast that a maximal single-turnover rate could not be measured using rapid mixing/quench techniques and is at least 850 s–1. The entire catalytic cycle is limited by a slow step that follows chemistry and generates a steady-state incision rate of about 2 s–1. Site-directed mutation of His-309 to Asn and Asp-210 to Ala reduced the single turnover rate of incision 5 ′ to AP sites by at least 5 orders of magnitude such that chemistry (or a step following DNA binding and preceding chemistry) and not a step following chemistry became rate-limiting. Our results suggest that the efficiency with which APE1 can process an AP site in vivo is limited by the rate at which it diffuses to the site and that a slow step after chemistry may prevent APE1 from leaving the site of damage before the next enzyme arrives to continue the repair process. Human AP endonuclease 1 (APE1, REF1) functions within the base excision repair pathway by catalyzing the hydrolysis of the phosphodiester bond 5 ′ to a baseless sugar (apurinic or apyrimidinic site). The AP endonuclease activity of this enzyme and two active site mutants were characterized using equilibrium binding and pre-steady-state kinetic techniques. Wild-type APE1 is a remarkably potent endonuclease and highly efficient enzyme. Incision 5 ′ to AP sites is so fast that a maximal single-turnover rate could not be measured using rapid mixing/quench techniques and is at least 850 s–1. The entire catalytic cycle is limited by a slow step that follows chemistry and generates a steady-state incision rate of about 2 s–1. Site-directed mutation of His-309 to Asn and Asp-210 to Ala reduced the single turnover rate of incision 5 ′ to AP sites by at least 5 orders of magnitude such that chemistry (or a step following DNA binding and preceding chemistry) and not a step following chemistry became rate-limiting. Our results suggest that the efficiency with which APE1 can process an AP site in vivo is limited by the rate at which it diffuses to the site and that a slow step after chemistry may prevent APE1 from leaving the site of damage before the next enzyme arrives to continue the repair process. Human apurinic/apyrimidinic endonuclease 1 (APE1) 2The abbreviations used are: AP, apurinic, apyrimidinic, or abasic; APE1, AP endonuclease 1; BER, base excision repair; RhX, X-rhodamine; WT, wild-type; EP, enzyme-product; ES, enzyme-substrate. is a multifunctional protein that is required for cell viability. APE1 is an endonuclease that functions within the base excision repair (BER) pathway to initiate the repair of abasic (AP) sites in DNA (reviewed in Refs. 1Wilson III, D.M. Barsky D. Mutat. Res. 2001; 485: 283-307Crossref PubMed Scopus (341) Google Scholar and 2Evans A.R. Limp-Foster M. Kelley M.R. Mutat. Res. 2000; 461: 83-108Crossref PubMed Scopus (502) Google Scholar). The BER pathway is a series of enzymatically catalyzed reactions used by cells to remove and replace damaged or lost bases (reviewed in Ref. 3Krokan H.E. Nilsen H. Skorpen F. Otterlei M. Slupphaug G. FEBS Lett. 2000; 476: 73-77Crossref PubMed Scopus (311) Google Scholar). APE1 catalyzes the hydrolysis of the sugar-phosphate backbone of DNA 5′ to an AP site. The 3′ end of the nick generated in the damaged DNA strand can then be extended by a DNA polymerase to replace the damaged or missing base. Human APE1 is homologous to Escherichia coli EXOIII (4Demple B. Herman T. Chen D.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11450-11454Crossref PubMed Scopus (478) Google Scholar, 5Robson C.N. Hickson I.D. Nucleic Acids Res. 1991; 19: 5519-5523Crossref PubMed Scopus (293) Google Scholar), and like EXOIII, it possesses 3′-5′-exonuclease activity as well as 3′-phosphatase and 3′-phosphodiesterase activities (6Seki S. Hatsushika M. Watanabe S. Akiyama K. Nagao K. Tsutsui K. Biochim. Biophys. Acta. 1992; 1131: 287-299Crossref PubMed Scopus (112) Google Scholar). Unlike E. coli EXOIII, the AP endonuclease activity of human APE1 is far more robust than any of its other repair activities (7Kane C.M. Linn S. J. Biol. Chem. 1981; 256: 3405-3414Abstract Full Text PDF PubMed Google Scholar). APE1 also has several other functions in the cell beyond its DNA repair functions and has been shown to stimulate the DNA binding activity of certain transcription factors (AP-1 (FOS/JUN), Egr-1, NF-κb, and p53). APE1 can act as a transcription factor itself and bind to promoters containing negative Ca2+-response elements (8Bhakat K.K. Izumi T. Yang S.H. Hazra T.K. Mitra S. EMBO J. 2003; 22: 6299-6309Crossref PubMed Scopus (159) Google Scholar, 9Kuninger D.T. Izumi T. Papaconstantinou J. Mitra S. Nucleic Acids Res. 2002; 30: 823-829Crossref PubMed Scopus (76) Google Scholar). Finally, a truncated form of APE1 with increased 3′-to 5′-exonuclease activity and increased endonuclease activity on undamaged DNA has been implicated in the fragmentation of the genome associated with apoptosis (10Yoshida A. Urasaki Y. Waltham M. Bergman A.C. Pourquier P. Rothwell D.G. Inuzuka M. Weinstein J.N. Ueda T. Appella E. Hickson I.D. Pommier Y. J. Biol. Chem. 2003; 278: 37768-37776Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 11Yoshida A. Pommier Y. Ueda T. Int. J. Hematol. 2006; 84: 31-37Crossref PubMed Google Scholar). All of the above functions of APE1 have been found to be essential for cell viability and/or cell division and differentiation (12Izumi T. Brown D.B. Naidu C.V. Bhakat K.K. Macinnes M.A. Saito H. Chen D.J. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5739-5743Crossref PubMed Scopus (187) Google Scholar, 13Zou G.M. Luo M.H. Reed A. Kelley M.R. Yoder M.C. Blood. 2007; 109: 1917-1922Crossref PubMed Scopus (76) Google Scholar). APE1-null mice or mouse embryonic stem cells could not be made because these embryos had significant developmental flaws and lost viability at about embryonic day 5.5 (14Xanthoudakis S. Smeyne R.J. Wallace J.D. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8919-8923Crossref PubMed Scopus (441) Google Scholar). Studies using RNA interference to reduce the levels of APE1 in several human cell types have shown that reduction of APE1 expression halted cell proliferation, resulted in an accumulation of AP sites in DNA, and stimulated cell death via apoptosis (15Fung H. Demple B. Mol. Cell. 2005; 17: 463-470Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Although the role of APE1 in base excision repair has been fairly well defined, the mechanism by which APE1 catalyzes the hydrolysis of the phosphodiester bond 5′ to an AP site has not been characterized as extensively. Questions as simple as how fast the hydrolysis reaction is remain to be answered. Presumably, hydrolysis of the phosphodiester bond requires mechanisms for 1) activating a water molecule or perhaps another nucleophile for attack on the phosphoryl group, 2) stabilizing a charge that builds up on what is most likely a pentameric phosphoryl transition state, and 3) donating a proton to the leaving group (3′-alkoxide). Divalent cations could perform some of these functions and are required for the AP endonuclease activity of APE1, but the number required and their role in catalysis is not clear. Depending on the pH, crystalline forms of the enzyme contain either one or two divalent cations in the active site (16Mol C.D. Izumi T. Mitra S. Tainer J.A. Nature. 2000; 403: 451-456Crossref PubMed Scopus (616) Google Scholar, 17Beernink P.T. Segelke B.W. Hadi M.Z. Erzberger J.P. Wilson III, D.M. Rupp B. J. Mol. Biol. 2001; 307: 1023-1034Crossref PubMed Scopus (164) Google Scholar, 18Gorman M.A. Morera S. Rothwell D.G. de La Fortelle E. Mol C.D. Tainer J.A. Hickson I.D. Freemont P.S. EMBO J. 1997; 16: 6548-6558Crossref PubMed Scopus (289) Google Scholar). Amino acid residues serving as general acids and/or general bases could also perform these functions in catalysis, and structural and mutagenesis studies have suggested that His-309 and Asp-210 could function in this capacity. His-309 has been proposed to hydrogen-bond to an oxygen on the phosphoryl moiety to stabilize charge in the transition state, and mutation of His-309 to Asn reduces steady-state AP endonuclease rates by 30,000-fold (19Lowry D.F. Hoyt D.W. Khazi F.A. Bagu J. Lindsey A.G. Wilson III, D.M. J. Mol. Biol. 2003; 329: 311-322Crossref PubMed Scopus (35) Google Scholar, 20Lucas J.A. Masuda Y. Bennett R.A. Strauss N.S. Strauss P.R. Biochemistry. 1999; 38: 4958-4964Crossref PubMed Scopus (43) Google Scholar). Two roles for Asp-210 have been proposed. First, it may activate a water molecule for attack on the phosphoryl, and second, it may subsequently protonate the 3′-alkoxide leaving group. Mutation of Asp-210 to Ala reduces the steady-state AP endonuclease activity by 25,000-fold (21Erzberger J.P. Wilson III, D.M. J. Mol. Biol. 1999; 290: 447-457Crossref PubMed Scopus (109) Google Scholar, 22Rothwell D.G. Hang B. Gorman M.A. Freemont P.S. Singer B. Hickson I.D. Nucleic Acids Res. 2000; 28: 2207-2213Crossref PubMed Scopus (53) Google Scholar). As an initial step in defining the kinetic mechanism of APE1 and the contributions of two amino acid residues, His-309 and Asp-210, to catalysis, pre-steady-state kinetics of AP site incision and equilibrium binding to AP site-containing DNA were measured for wild-type and mutant enzymes. Preparation of 32P-labeled DNA Substrates and APE1 Proteins—All DNA substrates were of the sequence 5′-GCGTCAAAATGTFGGTATTTCCATG-3′, in which F indicates the position of tetrahydrofuran, a reduced abasic site analog. DNA duplexes were labeled on the 5′ end of the damaged strand with 32P and annealed to a complementary oligonucleotide containing an thymine opposite tetrahydrofuran as in Refs. 23Abner C.W. Lau A.Y. Ellenberger T. Bloom L.B. J. Biol. Chem. 2001; 276: 13379-13387Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar and 24Maher R.L. Vallur A.C. Feller J.A. Bloom L.B. DNA Repair (Amst.). 2007; 6: 71-81Crossref PubMed Scopus (16) Google Scholar. Mutant and WT APE1 proteins were expressed in and purified from E. coli as described previously (24Maher R.L. Vallur A.C. Feller J.A. Bloom L.B. DNA Repair (Amst.). 2007; 6: 71-81Crossref PubMed Scopus (16) Google Scholar). APE1 Site-directed Mutagenesis—Site-directed mutagenesis was used to generate APE1 mutants with specific amino acid changes in the active site. The pET-14b plasmid containing the APE1 sequence was mutagenized using the QuikChange (Stratagene) site-directed mutagenesis kit following the manufacturer's protocol. Histidine 309 was replaced with asparagine (H309N) using two primers of the sequence 5′-ccc tcg gca gtg ata act gtc cta tca ccc t-3′ and 5′-agg gtg ata gga cag tta tca ctg ccg agg g-3′ (Integrated DNA Technologies). Aspartic acid 210 was replaced with alanine (D210A) using two primers of the sequence 5′-cct tgt gct gtg tgg agc act caa tgt ggc aca tg-3′ and 5′-cat gtg cca cat tga gtg ctc cac aca gca caa gg-3′ (Integrated DNA Technologies). Fluorescence Anisotropy Binding Assays—An oligonucleotide 25 nucleotides in length of the sequence 5′-GCGTCAAAATGTFGGTATTTCCATG-3′ containing tetrahydrofuran, a reduced abasic site analog, indicated by F at position 13, was annealed to a complementary strand that contains a T opposite the AP site. This complement was labeled with X-rhodamine via an amino linker on the 5 position of a thymine two nucleotides from the 5′ end. These two strands were mixed in equal molar amounts in 50 mm HEPES, 100 mm KCl, 5% (v/v) glycerol, heated to 80 °C, and annealed through slow cooling to room temperature. Anisotropy measurements were obtained using a QuantaMaster QM-1 fluorometer (Photon Technology International) equipped with a 75-watt xenon arc lamp, an excitation monochromator, and dual emission monochromators. The excitation monochromator was set to 580 nm with a band pass of 8 nm leading to the cuvette. The dual monochromators, set in line with the photomultiplies tubes, were set to 610 nm with a band pass of 8 nm leading to the photomultiplies tube. All binding reactions had a final volume of 80 μl. Labeled substrate was diluted into the cuvette in binding buffer (50 mm HEPES pH 8, 100 mm KCl, 5% glycerol, 20 mm EDTA) to a final concentration of 20 or 40 nm. Anisotropy of the free DNA was calculated according to Equation 1, in which Ivv is the intensity of the vertically polarized emission and Ivh is the intensity of the horizontally polarized emission when exciting with vertically polarized light, and Equation 2, in which g is a correction factor to account for differences in the efficiencies of the two detectors. The g factor was calculated from the intensities of vertically, Ihv, and horizontally, Ihh, polarized emission measured using horizontally polarized excitation light. APE1 was added in various concentrations, and the “bound” anisotropy was calculated just as for the free DNA. These data were then globally fit to Equation 3, using Prism (GraphPad software), to determine the Kd in which Kd is the dissociation constant, Et is the total enzyme concentration, Dt is the total DNA concentration, robs is the observed anisotropy, rb is the maximum anisotropy, and rf is the anisotropy of the unbound DNA. robs=Ivv−gIvhIvv+2gIvh(Eq. 1) g=IhvIhh(Eq. 2) robs=((Kd+Et+Dt)−((Kd+Et+Dt)2−4EtDt)2Dt)×(rb−rf)+rf(Eq. 3) Electrophoretic Mobility Shift Binding Assays—A 25-mer oligonucleotide containing tetrahydrofuran was labeled at the 5′ end with 32P as described above and annealed to a complementary strand with a thymine opposite tetrahydrofuran. This substrate was then mixed at room temperature with various amounts of APE1 in electrophoretic mobility shift assay buffer (50 mm HEPES pH 8, 100 mm KCl, 10% glycerol, 20 mm EDTA, 0.1 mg/ml bovine serum albumin). Aliquots of the binding mixtures were loaded into a 6% non-denaturing polyacrylamide gel. Polyacrylamide electrophoresis was performed at 4 °C at 8 V/cm2 for 90 min. Gels were visualized and quantified using the Storm PhosphorImager and ImageQuant analysis software (Amersham Biosciences). Data were fit to Equation 4 using SigmaPlot (Systat Software) to determine the Kdapp in which [ED] is the concentration of the DNA bound by the enzyme, Kdapp is the apparent dissociation constant, Et is the total enzyme concentration, and Dt is the total DNA concentration. ED]=(Kdapp+Et+Dt)−(Kdapp+Et+Dt)2−4EtDt)2(Eq. 4) APE1 AP Endonuclease Activity Assays—APE1 endonuclease activity assays were performed using the KinTek RQF-3 rapid quench instrument (KinTek) in constant quench mode. APE1 and tetrahydrofuran containing DNA were mixed in a reaction buffer containing 50 mm HEPES pH 8, 100 mm KCl, 5% (v/v) glycerol, 5 mm MgCl2, and 0.2 mg/ml bovine serum albumin. Reactions were quenched using 0.2 m NaOH. Two volumes of 96% formamide, 20 mm EDTA were added to quenched reactions, which were then heated to 95 °C for 10 min. Substrates were separated from products by denaturing PAGE on 12% acrylamide, 8 m urea gels. Gels were visualized and quantified using a Storm PhosphorImager and ImageQuant analysis software (Amersham Biosciences). The concentration of sodium hydroxide necessary to effectively quench the reaction was determined empirically by titrating the quencher. In one series of experiments done in duplicate, reactions containing 20 nm AP-DNA and 1,000 nm APE1 were quenched after 2 ms with 0, 0.05, 0.1, 0.2, 0.3, and 0.4 m NaOH. In the absence of quencher, substrate was completely converted to product (98.8%), whereas the sodium hydroxide quencher stopped reactions with about the same efficiency at all concentrations tested. Concentrations of 0.05 and 0.1 m NaOH limited the percentage of substrate converted to product to 80.8 ± 0.2%, and concentrations of 0.2, 0.3, and 0.4 m NaOH limited product formation to 79.3 ± 0.5%. AP endonuclease activity assays for the APE1 mutants were done using hand mixing techniques with the same buffers and conditions used in the assays for the wild-type enzyme. Multiple-turnover assays for wild-type APE1 were fit by single exponentials plus linear steady-state phases (Equation 5). Single-turnover data for wild-type APE1 were fit to double exponentials (Equation 6), and single-turnover data for APE1 mutants were fit to single exponentials (Equation 7). Curve fitting was done using SigmaPlot (Systat Software), and figures were made using Kaleidagraph (Synergy Software). y=a(1−e−kt)+st(Eq. 5) y=a(1−e−k1t)+b(1−e−k2t)(Eq. 6) y=a(1−e−kt)(Eq. 7) Equilibrium Binding to AP Site DNA—A fluorescence anisotropy-based binding assay was employed to determine the binding affinity of APE1 for DNA duplexes containing an AP site (AP-DNA). Duplex DNA contained a single reduced abasic site analog, tetrahydrofuran, at position 13. APE1 binds both natural and reduced AP sites and catalyzes hydrolysis of the 5′-phosphodiester bond (25Wilson III, D.M. Takeshita M. Grollman A.P. Demple B. J. Biol. Chem. 1995; 270: 16002-16007Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The reduced AP site was used in these assays because it is less reactive and more stable than the natural AP site. Duplexes were covalently labeled with X-rhodamine (RhX) via an amino linker on the 5-position of a thymine located on the strand complementary to the AP-containing strand, two nucleotides from the 5′ end. Binding of WT APE1 to AP-DNA was determined by measuring the anisotropy of the RhX probe in assays containing either 20 or 40 nm AP-DNA in the presence of 20 mm EDTA to inhibit strand incision. Given that the APE1-AP-DNA complex is larger and tumbles more slowly than free AP-DNA, the anisotropy of the RhX reporter increases as the fraction of bound DNA increases (26Lakowicz J.R. Principles of Fluorescence Spectroscopy. Second. Kluwer Academic/Plenum, New York1999Crossref Google Scholar). Binding data from four independent experiments were globally fit to a single dissociation constant (Equation 3) yielding a Kd value of 11 ± 2nm for WT APE1 (Fig. 1). Binding to undamaged DNA was not detectable under conditions used in Fig. 1 (data not shown), and therefore, this Kd value reflects a specific interaction with AP-DNA. Multiple-turnover AP Site Incision Assays—Incision 5′ to a reduced AP site by APE1 was measured under pre-steady-state multiple-turnover conditions in which AP-DNA was present in excess over enzyme. AP endonuclease reactions were initiated by the rapid addition of APE1 to 32P-labeled DNA as described under “Experimental Procedures.” Time courses for product formation were measured by rapidly quenching reactions in sodium hydroxide after different incubation times, separating DNA substrates from products by denaturing PAGE and phosphorimaging. Two series of reactions were done. One series (Fig. 2, upper panel) contained 40 nm APE1 and 80, 160, 250, 350, or 500 nm AP-DNA, and the second series (Fig. 2, lower panel) contained 200 nm APE1 and 500, 1,000, or 2,000 nm AP-DNA. Time courses for both series of reactions gave a rapid burst of product followed by a linear increase in the amount of product. This kinetic behavior shows that the first round of conversion of substrate to product occurs rapidly but that some step following the chemical step limits the rate at which APE1 can dissociate from product and catalyze subsequent rounds of incision. The overall rate of product formation in the rapid pre-steady-state phase of the reaction increased with increasing DNA concentrations. However, due to difficulty in quantitating the relatively small fraction of substrate converted to product in the burst phase of the reactions, particularly at high DNA concentrations, rates obtained from empirical fits of these data (Fig. 2, solid curves) to an exponential plus a linear rise (Equation 5) contain considerable error. That being said, these fits indicated that the pre-steady-state burst rate was greater than 200 s–1 in reactions containing 200 nm APE1 and 500 nm AP-DNA (legend for Fig. 2). Steady-state rate constants for the linear phase of product formation were 1.4 ± 0.1 and 2.3 ± 0.4 s–1 for experiments done at 40 and 200 nm APE1, respectively, or about 2 orders of magnitude slower than the observed rate for the pre-steady-state burst. Another notable feature of both series of reactions was that the burst amplitudes were less than one enzyme equivalent of product. The amplitudes for reactions containing 40 nm APE1 are just under 30 nm, and the amplitudes for reactions containing 200 nm APE1 are less than 150 nm. One explanation for burst amplitudes that are less than one enzyme equivalent in multiple-turnover reactions is that the total protein concentration determined from the absorbance at 280 nm (see “Experimental Procedures”) is greater than the concentration of active enzyme. APE1-AP-DNA Binding under Stoichiometric Conditions–To test possibility that the active site concentration of APE1 was less than the total protein concentration, binding of APE1 to AP-DNA was measured under stoichiometric conditions using the RhX anisotropy-based assay. Binding reactions contained 1,000 nm AP-DNA in the presence of 20 mm EDTA to inhibit the incision reaction. Fig. 3 shows the average results for two independent experiments. The increase in anisotropy due to APE1-AP-DNA binding reaches a maximum at a concentration of about 1,000 nm APE1 or an enzyme:DNA ratio of about 1:1 and does not increase further. The simplest interpretation of this result is that the enzyme is at least 90% active, and the enzyme binds AP-DNA as a monomer. This result suggests that the reduced burst amplitudes in multiple-turnover incision reactions may not stem from a reduction in the active site concentration relative to the total protein concentration. Single-turnover AP Site Incision Assays—Another explanation for burst amplitudes less than one enzyme equivalent in multiple-turnover experiments is an internal equilibrium between enzyme-substrate and enzyme-product complexes that arises from a significant rate of the reverse reaction relative to the forward reaction (27Johnson K.A. The Enzymes. 1992; 20: 1-60Crossref Scopus (379) Google Scholar). To test this possibility and to determine the maximal rate of incision 5′ to AP sites, the AP endonuclease reaction was measured under single-turnover conditions with enzyme in excess of DNA. Reactions contained 0.02 μm AP-DNA and APE1 concentrations ranging from 0.04 to 6 μm (Fig. 4) and were initiated by the rapid addition of AP-DNA to APE1. The overall rates of the AP endonuclease reactions increased with increasing enzyme concentrations; however, analysis of individual reaction progress curves showed that each was composed of two phases and was best fit by a double exponential rise to a maximum (Equation 6 and Fig. 4, solid curves through the data points). Data from a second independent series of single-turnover experiments gave the same results and are presented separately in supplemental Fig. 1 for clarity. The rate of the rapid phase increased linearly with enzyme concentration until a concentration of about 2 μm APE1, at which point rates became too fast to measure experimentally (Figs. 4 and 5). Given that the observed rates are a function the rates of both enzyme-DNA binding and formation of product in the enzyme active site (Fig. 5, steps a and b in the reaction scheme), a linear increase in rate with enzyme concentration indicates that enzyme-DNA binding and not chemistry is rate-limiting over this concentration range. A fit of the observed rates of the rapid phase as a function of enzyme concentration to a line (Fig. 5) yielded an apparent on-rate constant (slope) of 3.5 × 108 m–1 s–1 and an apparent off-rate constant (y intercept) 47 s–1 (27Johnson K.A. The Enzymes. 1992; 20: 1-60Crossref Scopus (379) Google Scholar). At concentrations greater than 2 μm APE1, about 70% of the substrate was consumed in the shortest time, 2 ms, that could be measured, and the majority of the rapid phase was already complete. This rapid reaction precluded determination of a maximal single-turnover rate, but it must be at least 850 s–1 to give rise to these data.FIGURE 5Plot of observed rate constants from fits of single-turnover data as a function of WT APE1 concentration. Observed rate constants calculated for the rapid phase of single-turnover data plotted in Fig. 4 and supplemental Fig. 1 were plotted as a function of APE1 concentration and fit (solid line) to a line. At concentrations greater than 2 μm APE1, accurate values for this observed rate constant could not be determined because the reactions were too fast. An apparent binding constant (kon,app of 3.5 × 108 m–1 s–1 and apparent dissociation constant (koff,app) of 47 s–1 were calculated from the slope and y intercept, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The amplitudes of each kinetic phase were also a function of enzyme concentration. At the lowest APE1 concentration, 0.04 μm, the amplitudes for the rapid and slow phases were about the same. As the APE1 concentration increased, the fraction of product turned over in the rapid phase, as measured by the amplitude, increased, whereas the amplitude of the slow phase decreased (Fig. 4 and supplemental Fig. 1). At APE1 concentrations greater than 2 μm, the amplitudes no longer changed with increasing APE1 concentrations and reached maximum values of about 16 nm product formed in the rapid phase and 4nm in the slow phase for reactions that contained 20 nm AP-DNA substrate. The biphasic nature of the single-turnover kinetic data is consistent with an internal equilibrium being established between enzyme-substrate and enzyme-product that requires an “irreversible” step to drive the reaction toward completion. The fraction of product formed in the rapid phase reflects product produced in the enzyme active site ((enzyme-product) EP), and the slow phase reflects the step that drives the pathway forward by limiting the reverse reaction. The observed rates of the slow phase showed a smaller dependence on enzyme concentration and increased from a value of about 4 s–1 at 0.4 μm APE1 to a maximum value of about 20–30 s–1 at APE1 concentrations of 0.5 μm and greater. Effects of Mutation of His-309 to Asn on APE1 DNA Binding and AP Endonuclease Activity—Binding of APE1-H309N to the same AP-DNA substrate used for WT APE1 in Fig. 1 was measured using the anisotropy-based binding assay. A Kd value of 53 ± 7nm was calculated from data shown in Fig. 6A, a value about 5-fold greater than that for WT APE1. Single-turnover AP site incision assays were performed for APE1-H309N as described for those with WT APE1 (Fig. 4); however, reactions catalyzed by the mutant were so slow that assays could be done by hand mixing rather than using a rapid-mixing apparatus. Results from experiments done using 20 nm AP-DNA and 100–1000 nm APE1-H309N are shown in Fig. 6B. In contrast to reactions with WT APE1, the rate of incision 5′ to the AP site reached a maximal value at saturating APE1-H309N concentrations. A fit of these data to a single exponential (Equation 7 and Fig. 6B, solid lines through data) yielded a maximal rate of 0.001 s–1. Effects of Mutation of Asp-210 to Ala on APE1 DNA Binding and AP Endonuclease Activity—Binding of APE1-D210A to AP-DNA was measured in the anisotropy-based equilibrium binding assay. A Kd value of 2.4 ± 1.3 nm was calculated from the binding isotherm shown in Fig. 7A. Given that the Kd value was about 10-fold lower than the absolute DNA concentration used in the binding assay, it was desirable to repeat the measurement at a lower DNA concentration to confirm this value. Because the fluorescence at low DNA concentrations was relatively weak, these experiments were done using 32P-labeled AP-DNA in an electrophoretic mobility shift assay. Radiolabeled AP-DNA (5 nm) was incubated with APE1-D210A at concentrations ranging from 1 to 20 nm in the presence of EDTA. Bound DNA was separated from free DNA by electrophoresis on non-denaturing polyacrylamide gels and quantitated by phosphorimaging. A fit of these data (Fig. 7B) to a quadratic equation (Equation 4) yielded a Kd value of 1.5 ± 0.4 nm in good agreement with the value of 2.4 ± 1.3 nm obtained from the anisotropy-based assay. These results show that APE1-D210A binds AP-DNA with about 5-fold greater affinity than WT APE1 and about 25-fold greater than APE1-H309N. AP endonuclease activity of APE1-D210A was measured in single-turnover assays as done for APE1-H309N in reactions containing 20 nm AP-DNA and up to 20 μm APE1-D210A. In the case of the D210A mutant, the incision reaction was much slower, and less than half the substrate was converted to product in 23 h (data not shown). Rates were not calculated based on these experiments because of the prolonged incubation times required and the possibility that loss of enzyme activity during the extended reaction could contribute to the kinetics. APE1 is the major enzyme in human cells responsible for initiating the repair of AP sites in DNA. Although much work has been done in characterizing the AP endonuclease activity under steady-state conditions, this is the first study to investigate the pre-steady-state kinetics of incision 5′ to AP sites by APE1. This work showed that the first turnover of substrate by APE1 is rapid" @default.
• W2044974504 created "2016-06-24" @default.
• W2044974504 creator A5001509910 @default.
• W2044974504 creator A5002499771 @default.
• W2044974504 date "2007-10-01" @default.
• W2044974504 modified "2023-09-29" @default.
• W2044974504 title "Pre-steady-state Kinetic Characterization of the AP Endonuclease Activity of Human AP Endonuclease 1" @default.
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